Mechanistic understanding of HIV-1 broad neutralizing antibody evolution Recent advances in animal experiments and clinical trials using broad neutralizing antibodies (bnAbs) as prophylactic and therapeutic agents hold a great promise for more effective treatments of HIV/AIDS. In order to make the passive immunization become a real therapy, new bnAbs should be isolated and their activities should be characterized at molecular levels. However, the mechanism underlying the development of bnAbs is largely unknown and there is no vaccination regimen known to elicit bnAb effectively in vivo. BnAbs are developed in a subset of human patients after years of chronic HIV-1 infection, which exhibit great potency and breadth of the neutralizing activities. Up to date, all HIV-1 bnAbs were directly isolated from patients, which is costly, time-consuming, and completely by chance. The goal of this project is to establish an in vitro mutagenesis platform to derive new HIV-1 bnAbs, and to delineate the stepwise accumulations of mutations critical for the bnAb maturation. For most bnAbs, their germline precursors do not bind to native HIV-1 epitopes, or bind very poorly. Finding the proper antigens to stimulate the initial or intermediate precursor B cells remains a daunting task. Without proper stimulation, precursor B cells do not enter the germinal center reaction necessary for acquiring somatic hypermutations (SHM) for affinity maturation. Although the affinity selection process in germinal center is difficult to recapitulate in vitro, several human B cell lines are capable of constitutive or inducible SHM. Therefore, for the purpose of antibody diversification, mutating V regions in a hypermutating human B cell line is as good as in germinal center B cells. It can be envisioned that knock-in of bnAb progenitor genes into a hypermutating human B cell line (Ramos) can give rise to a large pool of mutants from which new bnAbs can be derived. Once this in vitro system is established, we will test two hypotheses: i) bnAbs can be evolved in vitro by mutagenizing their germline or intermediate precursors; ii) the potency and breadth of bnAbs can be further increased by SHM in vitro. The expected outcome of this project is to establish a new method of generating therapeutic bnAbs against HIV-1 (and other viruses), and a new platform for mechanistic study of the evolution of mutations critical for broad neutralizing activities. The Scientific Premise of this project is based on the promising testing results of HIV-1 bnAbs in preclinical and clinical experiments and the extensive studies of SHM in the Ramos cell line.